Epigenetic regulation is critical to diverse biological processes and diseases. Post-translational modifications (PTMs) in histones are generally considered to be a major group of epigenetic marks, contributing to the epigenetic program that dictates diverse DNA-templated biological outputs. Our team has recently identified nine types of novel histone PTMs (lysine propionylation, lysine butyrylation, lysine malonylation, lysine succinylation, lysine glutarylation, lysine crotonylation, lysine 2-hydroxyisobutyrylation, lysine 3-hydroxybutyrylation, and lysine benzoylation). Importantly, we and others demonstrated critical roles for these new PTM pathways by revealing their unique functions in the transcriptional control of gene expression and cellular regulation, and by linking them to multiple inborn metabolic diseases. Some of these histone marks can be stimulated by cellular levels of their corresponding short-chain lipids, thus suggesting new mechanisms for metabolism-regulated epigenetic changes. This application is based on the tremendous expertise we gained during our earlier studies on identification and characterization of nine novel types of histone marks. We hypothesize that additional, undescribed types of histone PTMs are present in mammalian histones. Our hypothesis is supported by our preliminary data identifying multiple undescribed PTMs in histones, the chemical structures of which are either undefined or remain to be fully validated. We propose to use an integrated approach, employing technologies that have been well established and fully validated in the laboratories of our team, to comprehensively screen for novel types of histone PTMs. Our team is best positioned to find these histone marks because of the tremendous skills and numerous technologies we have accumulated, and the high productivity we have demonstrated in our recent identification of the novel types of histone marks. We will first comprehensively screen for new types of histone marks and validate them by both chemical and biochemical methods. We will then investigate their epigenetic roles during spermatogenesis. New types of histone marks will significantly advance our understanding of chromatin structure and function, might open unforeseen avenues for epigenetic studies, and may even shift the focus of current research on epigenetic mechanisms and disease.